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J. Med. Chem. 2004, 47, 5244-5250
Rational Design, Synthesis, and Biological Evaluation of Bis(pyrimido[5,6,1-de]acridines) and Bis(pyrazolo[3,4,5-kl]acridine-5-carboxamides) as New Anticancer Agents Ippolito Antonini,* Paolo Polucci,† Amelia Magnano, Silvia Sparapani, and Sante Martelli Department of Chemical Sciences, University of Camerino, Via S. Agostino 1, 62032 Camerino, Italy Received April 20, 2004
The good results obtained with pyrimido[5,6,1-de]acridines 7 and with pyrazolo[3,4,5-kl]acridinecarboxamides 8 prompted us to the synthesis of two new series of bis acridine derivatives: the bis(pyrimidoacridines) 5 and the bis(pyrazoloacridinecarboxamides) 6. Compounds 5 can be regarded also as cyclized derivatives of bis(acridine-4-carboxamides) 3 and compounds 6 as cyclized derivatives of bis(acridine-4-carboxamides) 4. The noncovalent DNAbinding properties of these compounds have been examined using fluorometric techniques. The results indicate that (i) the target compounds are excellent DNA ligands; (ii) the bis derivatives 5 and 6 are more DNA-affinic than corresponding monomers 7 and 8; (iii) the new bis 5 and 6 result always less efficient in binding than related bis(acridine-4-carboxamides) 3 and 4; and (iv) in both series 5 and 6 a clear, remarkable in some cases, preference for binding to AT rich duplexes can be noted. In vitro cytotoxic potency of these derivatives toward the human colon adenocarcinoma cell line (HT29) is described and compared to that of reference drugs. Structure-activity relationships are discussed. We could identify six very potent cytotoxic compounds for further in vitro studies: a cytotoxic screening against six human cancer cell lines and the National Cancer Institute (NCI) screening on 60 human tumor cell lines. Finally, compound 6a was selected for evaluation in a NCI in vivo hollow fiber assay. Introduction In past years, interest in symmetric bifunctional intercalators has been growing and a number of derivatives, synthesized employing different chromophores, have been studied.1-10 The noticeable results in the field may be exemplified by LU 79553 (1, Figure 1) and WMC-26 (2, Figure 1), both showing high effectiveness against tumor xenografts in vivo.6,7 We have also described the synthesis and the biological properties of two novel interesting classes of antitumor agents belonging to this type of derivative: the bis(acridine-4carboxamides) 3 and 4 (Figure 2).11 From them, 3a [X ) H, Y ) (CH2)3N(Me)(CH2)3], 3d [X ) NO2, Y ) (CH2)3N(Me)(CH2)3], 4a [X ) H, Y ) (CH2)3N(Me)(CH2)3], and 4b [X ) OMe, Y ) (CH2)3N(Me)(CH2)3] emerged as lead derivatives.11 Compounds 3a,d and 4a,b have fulfilled our purpose to enhance the outstanding biological response shown by the corresponding bisfunctionalized acridone-4-carboxamide monomers. 12 Prompted by the above results, we designed the synthesis of two new classes of potential bis intercalators: the bis(pyrimidoacridines) 5 and the bis(pyrazoloacridinecarboxamides) 6, which can be regarded as cyclized derivatives of 3 and 4, respectively (Figure 2). On the other hand, the chromophore moiety of 5 and 6 being constituted by the pyrimido[5,6,1-de]acridine-1,3,7-trione and by the pyrazolo[3,4,5-kl]acridine, respectively (Figure 2), we expected also an increase of the notable * Address correspondence to this author. Tel: +390737402235. Fax: +390737637345. E-mail:
[email protected]. † Present address: Department of Chemistry, 75/B1, Discovery Research Oncology, Pharmacia Corporation, Viale Pasteur 10, 20014 Nerviano (MI), Italy.
Figure 1. Structures of LU 79553 (1) and WMC-26 (2).
antitumor properties of the corresponding monomers 713 and 8.14 Finally, we have better investigated the relevance of the linker Y for biological activity of these compounds. Chemistry Schemes 1 and 2 show the synthetic pathways leading to target derivatives 5 and 6. According to Scheme 1, the reaction of the suitable 6-chloro-2-[2-(dimethylamino)ethyl]pyrimido[5,6,1-de]acridine-1,3,7-trione (7)13a,b with the appropriate R,ω-diamine in ethoxyethanol, in the presence of triethylamine at 80 °C, afforded the target bis(pyrimidoacridines) 5a-j. All the diamines were commercially available, except the N1,N2-bis(2aminoethyl)-N1,N2-dimethyl-1,2-ethanediamine, needed for 5g, that was prepared according to the literature.5 Cleavage with aqueous HBr of the methoxy derivatives 5h,i gave the hydroxy derivatives 5k,l, respectively. Target compound 5m could not be prepared in the same way, due to the difficulty in obtaining the 9-nitro
10.1021/jm049706k CCC: $27.50 © 2004 American Chemical Society Published on Web 09/11/2004
New Bis(acridine derivatives)
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5245
Figure 2. Rational design of 5 and 6 which can be regarded either as cyclized derivatives of the bis(acridine-4-carboxamides) 3 and 4 or as bis derivatives of the corresponding monomers 7 and 8.
Scheme 1a
a Reagents: (i) H N-Y-NH , N(Et) ; (ii) HBr 48%; (iii) COCl , N(Et) . Linker Y: (CH ) N(Me)(CH ) for 5a,h,j,k; (CH ) N(Me)(CH ) 2 2 3 2 3 2 3 2 3 2 2 2 2 for 5b,i,l; (CH2)3 for 5c; (CH2)6 for 5d; (CH2)8 for 5e; (CH2)12 for 5f; (CH2)2N(Me)(CH2)2N(Me)(CH2)2 for 5g. Substituents X: H for 5a-g; 9,9′-OCH3 for 5h,i; 9,9′,10,10′-OCH3 for 5j.
Scheme 2a
a Reagents: (i) H N-NH-(CH ) N(CH ) ; (ii) HBr 48%. Linker Y: (CH ) N(Me)(CH ) for 6a,c,e; (CH ) N(Me)(CH ) for 6b,d,f. 2 2 2 3 2 2 3 2 3 2 2 2 2 Substituents X: H for 6a,b; 9,9′-OMe for 6c,d.
derivative of 7.13b However, direct cyclization of 4b, performed in CHCl3 with COCl2 and triethylamine at room temperature, afforded 5m. As shown in Scheme 2, the bis(pyrazolo[3,4,5-kl]acridinecarboxamides) 6a-d were prepared by reaction
of the appropriate bis(acridine-4-carboxamides) 9a-d11 with [(2-hydrazino)ethyl]dimethylamine in 2-ethoxyethanol at 120 °C. The hydroxy derivatives 6e,f were obtained by refluxing the corresponding methoxy derivatives 6c,d in aqueous HBr.
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Table 1. Melting Points,a Yields, Formula,b DNA Binding,c and Cytotoxic Activity against Human Colon Adenocarcinoma (HT29) of Target Compounds 5a-m and 6a-f, of Related Bis(acridine-4-carboxamides) 3 and 4, of Corresponding Monomers 7 and 8, and of Mitoxantrone (Mx) compd
mp, °C
yield, %
5a 3ah 7ai 5b 5c 5d 5e 5f 5g 5h 3bh 7bi 5i 5j 7ci 5k 3ch 7di 5l 5m 3dh 7ei 6a 4ah 8aj 6b 6c 4bh 8bj 6d 6e 4ch 8cj 6f Mx
143-144 (259-260 d)
59
C45H49N9O6
183-184 (275-277 d) 222-224 (273-274) 275-277 (239-241) 242-244 (185-187) 187-189 (254-256) 182-184 (>350) 183-185 (258-259 d)
60 50 66 64 81 29 44
C43H45N9O6 C41H40N8O6 C44H46N8O6 C46H50N8O6 C50H58N8O6 C46H52N10O6 C47H53N9O8
200-202 (253-254 d) 190-192 (240-242 d)
48 30
C45H49N9O8 C49H57N9O10
263-264 (265-267 d)
95
C45H49N9O8
279-280 (270-272 d) 148-149 (262-263)
97 59
C43H45N9O8 C45H47N11O10
(>350)g
74
C43H51N11O2‚3HCl‚3H2O
(230-231)g (204-205)g
34 67
C41H47N11O2‚3HCl‚2H2O C45H55N11O4‚3HCl‚3H2O
(245-247)g 239-240 (260-261)
60 40
C43H51N11O4‚3HCl‚2H2O C43H51N11O4‚3HCl‚3H2O
282-283 (255-257)
39
C41H47N11O4‚3HCl‚2H2O
formula
Kappd × 10-7 M-1 AT CT-DNA GC 126 9.3 0.68 4.0 2.5 8.2 6.9 0.49 31 7.1 26 0.73 0.90 11 0.40 13 13 0.79 3.9 26 19 0.31 17 8.2 2.5 20 9.9 13 4.1 5.0 25 13 9.7 8.5
7.1 10 1.7 2.1 2.8 3.3 3.6 0.36 15 2.1 6.2 1.3 0.34 6.2 1.5 8.5 12 3.5 3.7 14 14 0.79 10 6.3 4.9 23 6.3 20 17.5 1.7 4.3 4.4 3.4 4.9 34
5.0 7.5 1.2 0.94 0.28 0.27 0.17 0.041 0.37 3.4 14 3.3 0.88 2.4 3.2 7.8 7.4 3.2 0.43 11 23 2.6 8.2 0.79 0.73 5.2 1.8 5.2 2.6 1.8 19 8.3 1.9 3.0
binding site preferencee
IC50 (nM)f HT29
A-T (25) none G-C (0.57) A-T (4.3) A-T (8.9) A-T (30) A-T (41) A-T (12) A-T (84) A-T (2.1) A-T (1.9) G-C (0.22) none A-T (4.6) G-C (0.12) A-T (1.7) A-T (1.8) G-C (0.25) A-T (9.1) A-T (2.4) none G-C (0.12) A-T (2.1) A-T (10) A-T (3.4) A-T (4.3) A-T (5.5) A-T (2.5) A-T (1.6) A-T (2.8) A-T (1.3) A-T (1.6) A-T (5.1) A-T (2.8)
9,9′,10,10′-OMe > 9,9′-OMe, and, similarly, for derivatives with Y ) (CH2)2N(CH3)(CH2)2, it is 9,9′-OH > 9,9′-H > 9,9′-OMe; in series 6 the behavior is different; for derivatives with Y ) (CH2)3N(CH3)(CH2)3, the rank is 9,9′-H > 9,9′-OMe > 9,9′-OH, whereas, for derivatives
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Journal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5247
with Y ) (CH2)2N(CH3)(CH2)2, the rank is 9,9′-H > 9,9′OH > 9,9′-OMe. (c) Regarding the chromophore: comparing the analogue pairs 5a,6a, 5b,6b, 5i,6d, and 5l,6f, the pyrazoloacridine chromophore seems more efficient than the pyrimidoacridone chromophore; only in the analogue pair 5k,6e we have an opposite trend. (d) Regarding the comparison with monomers: the bis derivatives 5 and 6, with Y ) (CH2)3N(CH3)(CH2)3, result always more efficient than corresponding monomers 7 and 8, with the only exception of 6c and 8b. (e) Regarding the comparison with related bis(acridine-4carboxamides): the new bis derivatives 5 and 6 result always less efficient than related bis(acridine-4-carboxamides) 3 and 4, with the only exception of 6a and 4a. Generally, the binding behavior of target compounds with synthetic polynucleotides reflects what we observed for CT-DNA. However, a clear, remarkable in some cases, preference for binding to AT rich duplexes is to be noted in both series 5 and 6. In contrast, compounds related to bis derivatives 5 have previously shown a decided GC preference (monomers 7) or a borderline AT preference (dimers 3), whereas compounds 6 show a binding site preference very similar to that of related bis derivatives 4 and monomers 8. Cytotoxic Activity. The target compounds 5a-m and 6a-f, the related bis 3 and 4, the corresponding monomers 7 and 8, and the reference drug mitoxantrone (Mx) were examined for antiproliferative activity against the human colon adenocarcinoma cell line (HT29). The results shown in Table 1 indicate that compounds 5a,h,k,m and 6a,c possess very potent antiproliferative activity, with IC50 values in the low/sub nM range, being remarkably more potent than Mx itself. The following remarks can be made: (i) Regarding the linker: (a) In the unsubstituted (X ) H) sub series 5a-g, with seven different linkers, 5a (Y ) (CH2)3N(CH3)(CH2)3) appears to be the most potent derivative, with IC50 value < 0.1 nM (at least 3 orders of magnitude lowest in the sub series), but also 5d (Y ) (CH2)6) is very active (IC50 43 nM); on the opposite site is 5f (Y ) (CH2)12) with the highest IC50 (3.5 µM); the other derivatives possess similar activity (IC50 range 0.11-0.39 µM). It seems that compounds 5a and 5d (Y ) (CH2)3N(CH3)(CH2)3 and Y ) (CH2)12, respectively) ensure an optimal linker length, but also the presence of a basic nitrogen atom, compound 5a, is important for cytotoxicity. (b) Comparison of the homologous pairs (5a,b, 5h,i, 5k,l, 6a,b, 6c,d, and 6e,f) clearly indicates that the best results are always obtained with Y ) (CH2)3N(CH3)(CH2)3 for both series 5 and 6, according to what is observed for related bis derivatives 3 and 4.11 The difference in potency is 3 orders of magnitude, at least, in series 5 and 2 orders of magnitude in series 6 for derivatives with Y ) (CH2)3N(CH3)(CH2)3. (ii) Regarding the substituents X: (a) In the series 6 the cytotoxicity rank order of derivatives with the same linker is 6c,d (X ) 9,9′-OMe) = 6a,b (X ) H) . 6e,f (X ) 9,9′-OH). (b) The nature of substituents in 9,9′ positions in the series 5 does not influence the activitiy too much. In the sub series with Y ) (CH2)3N(CH3)(CH2)3, all the derivatives 5a,h,k,m, possess very potent cytotoxicity (IC50 < 0.1 nM); in the sub series with Y ) (CH2)2N(CH3)(CH2)2, the derivatives 5b,i,l possess IC50
in the range 0.11-0.72 µM, with a weak influence giving the rank order 5b (X ) H) > 5l (X ) OH) > 5i (X ) OMe). (c) The only derivative with substituents in 9,9′,10,10′ positions, 5j (Y ) (CH2)3N(CH3)(CH2)3, X ) 9,9′,10,10′-OMe), possesses IC50 ) 0.25 µM, indicating that this kind of substitution is not detrimental for binding, but greatly diminishes cytotoxic potency. (iii) Regarding the chromophore: Comparing IC50 values of the pairs with Y ) (CH2)3N(CH3)(CH2)3 and the same X, 5a,6a, 5h,6c, and 5k,6e, it can be underlined that series 5 (pyrimido[5,6,1-de]acridine chromophore) is much more cytotoxic than series 6 (pyrazolo[3,4,5-kl]acridine chromophore), especially considering the pair 5k,6e. The results are in agreement with what is observed with the corresponding monomers 7 and 8. (iv) Finally, it should be noted that the target derivatives with Y ) (CH2)3N(CH3)(CH2)3, 5a, 5h, 5j, 5k, 5m, 6a, 6c, and 6e, are all more potent cytotoxic agents than the corresponding monomers 7 and 8; they are more potent than or equally potent to related bis derivatives 3 and 4. Generally, there is not a great correlation between IC50 and Kapp values. However, some considerations can be made: (a) In the homologous pairs of series 5, the linker Y ) (CH2)2N(CH3)(CH2)2 corresponds always to an inferior cytotoxicity and DNA affinity with respect to the linker Y ) (CH2)3N(CH3)(CH2)3. (b) Also in the homologous pairs of series 6, the shortest linker, Y ) (CH2)2N(CH3)(CH2)2, corresponds always to an inferior cytotoxicity with respect to the longest linker, Y ) (CH2)3N(CH3)(CH2)3, but for the DNA affinity the trend is different. (c) Compounds 5f,i, the weakest DNA ligands, are scarcely cytotoxic, but also compounds 5g,6b, the most potent DNA ligands, are scarcely cytotoxic; it can be deduced that DNA binding is not the only determinant for cytotoxic activity; other factors, e.g. cellular uptake, may influence the cytotoxicity. Compounds 5a,h,k,m, and 6a,c, the most potent in the series, were selected for a cytotoxic screening against six human cancer cell lines (large cell lung carcinoma H460M, gastric cancer MKN45, prostatic carcinoma PC3, colon adenocarcinoma HCT116, LoVo, sensitive, and LoVo/Dx, doxorubicin-resistant). The IC50 (nM) values after 1 and 144 h drug exposure are reported in Table 2. Reference drugs are Mx and doxorubicin (Dx). The results indicate the following: (i) The target derivatives are extremely potent cytoxic agents, especially compounds 5, which often present IC50 values inferior to the minimum drug concentration tested (10-3 nM). (ii) As previously noted with the HT29 cell line, compounds 5 are more potent than compounds 6, but here we can also discriminate among the potency of selected derivatives 5, in relation to the nature of substituents in 9,9′ positions. The rank order in potency seems to be 5k (X ) OH) > 5a (X ) H) > 5h (X ) OMe) > 5m (X ) NO2). (iii) Finally, it can be remarked that both derivatives 5 and 6 are cross resistant with Dx, but the grade of cross resistance of compounds 5 appear to be inferior to that of derivatives 6. Compounds 5a,h,k,m, and 6a,c, were also selected by the National Cancer Institute (NCI) for a screening on a panel of 60 human tumor cell lines. This screen is designed to discover spectrum of activity and, eventually, selectivity of drugs. The data from this assay can
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Table 2. Cytotoxic Screening against Six Human Cancer Cell Lines of Selected Compounds 5 and 6 after 1 h and 144 h of Drug Exposurea H460M 5a 5h 5k 5m 6a 6c Mx Dx a
MKN45
PC3
HCT116
LoVo/Dxb
LoVo
1h
144 h
1h
144 h
1h
144 h
1h
144 h
1h
144 h
1h
144 h
